Vaccine for prophylaxis of trichophytosis in horse and method of preparing same

ABSTRACT

The invention provides a vaccine for prophylaxis of trichophytosis in the horse which comprises a suspension of microconidia of the immunogenic strain Trichophyton equinum in a sterile physiological salt solution having a pH from 6.2 to 7.0, the concentration of the microconidia ranging from 30 to 45 million microconidia in 1 ml of sterile saline solution. The invention also provides a method for preparing said vaccine for prophylaxis of trichophytosis in the horse which involves growing the immunogenic culture Trichophyton equinum on a solid culture medium at a temperature of 26° to 28° C. for 15 to 25 days until optimum amounts of microconidia of said culture have accumulated, after which the resulting propagated biomass is separated, homogenized to prepare a suspension of separate microorganism cells, and the end product finally isolated by lyophilic drying. The use of the vaccine, prepared by the inventive method, rules out trichophytosis morbidity among horses to abolish losses due to untimely salts of pedigree and sporting horses, and also prevents infection of man. When tested on a mass scale, the vaccine proved highly efficacious (95-98%).

This is a continuation of application Ser. No. 719,976, filed Sept. 2,1976 now abandoned.

This invention relates to a new vaccine for prophylaxis oftrichophytosis in the horse and a method for preparing it.

Trichophytosis in the horse is a fungal infection caused by a species ofTrichophyton. The infection is transmitted by frequent contacts ofanimals belonging to different epizootic zones: hippodromes, sportsocieties, etc., that bring the animals together in numerous contests.

Trichophytosis-affected horses are not admitted to the sporting contestsand are withheld from sales. Treatment of the animals, disinfection andquarantine measures detain the animals at stud-farms and hippodromes.

Diseased animals are the source of infection of the personnel andsportsmen. The ever growing interest in equestrian sports increases thedanger of spreading the infection among the population.

Measures to control the disease are usually limited by traditional localtreatment of the affected sites of the skin with medical preparations,quarantines, disinfection, and also prophylactic treatment of the skin.The measures are time-consuming; yet they do not solve the problem ofeffective prophylaxis of trichophytosis in the horse.

The literature lacks information on the means of specific prophylaxis oftrichophytosis in the horse.

The new vaccine for prophylaxis of trichophytosis in the horsecomprises, according to the invention, a suspension of microconidia ofthe immunogenic strain Trichophyton equinum in a sterile saline solutionhaving a pH of 6.2 to 7.0, concentration of the microconidia rangingfrom 30 to 45 million microconidia in 1 ml of the sterile salinesolution. A saline solution, or physiological salt solution, is a 0.85%solution of sodium chloride in water. In addition to microconidia, thesuspension also contains fragments of mycelium and macroconidia. Allcalculations of concentration, vitality of the vaccine, etc., are madewith reference to the microconidia.

The vaccine is a white amorphous mass.

Vials with defective caps, as well as opened vials containing vaccinethat has not been used for vaccination, should be inactivated by boilingfor an hour. The preparation should be stored at a temperature from 4°to 12° C.

The vaccine is intended for prophylaxis of young and mature animals andis harmless. Active immunity to natural and artificial infectiondevelops in the vaccinated animals toward the end of a month followingvaccination. The immunity lasts for at least five years.

Vaccination of healthy animals does not cause trichophytosis in them.

According to the invention, the vaccine can be given intramuscularly forprophylaxis in a dose of 1 to 2 ml, in two injections, at a 10 to 14 dayinterval. The vaccine should be injected only into the third middle partof the neck. The injection site should be first disinfected.

In 7-15 days after the second vaccination, a localized crust, up to 15mm in diameter, is formed at the injection site. The crust isspontaneously rejected in 15 to 20 days.

Vaccination of the animals during the incubation period acceleratesdevelopment of the clinical manifestation of trichophytosis, but theanimals would normally recover without additional medicamentoustreatment.

Injection of double doses (compared with the prophylactic dose) todiseased animals, cures them in 15 to 25 days without any othertreatment.

When tried on a mass scale, the vaccine proved quite effective (95-98percent).

Prophylactic use of the vaccine practically prevents trichophytosismorbidity among horses; undue expenditures for materials and labour ofveterinary experts for multiple medicamentous treatment anddisinfection; failure in fulfillment of planned sales of pedigree andsports horses; extra keeping of trichophytosis-affected animals at studsand hippodromes; poor performance at competitions; and the danger ofinfecting humans.

The invention also includes a method of preparing the vaccine forprophylaxis of trichophytosis of the horse, which, according to theinvention, involves growing the immunogenic culture Trichophyton equinumon a solid nutrient medium, at a temperature of 26° to 28° C., for 15 to25 days, until optimum amounts of microconidia of said culture haveaccumulated in the medium. The procedure of determining microconidiaoptimal amount formation is as follows. A piece of the culture is takenfrom the matrices and placed on a slide glass. Thereafter upon addingone or two drops of distilled water thereto the piece is covered by acover glass. A preparation thus formed is looked through under themicroscope (×400).

There it must be at least 50-75 microconidia within a field of vision.

The resulting propagated biomass is then separated, homogenized toprepare a suspension of separate microorganism cells, the suspensionthus obtained is then dried lyophilically, and the end product isolated.

It is recommended that before lyophilic drying the concentration of theobtained suspension should be adjusted to 200 to 400 millionmicroconidia in 1 ml of a drying medium. The preferable composition ofthe drying medium is 35 to 45 percent by weight of saccharose and 5 to 7percent by weight of gelatin in distilled water.

The process for preparing the vaccine comprises the following stages:

inoculation, cultivation, and accumulation of the fungal mass of theculture Trichophyton equinum, homogenization of the fungal mass,standardization, filling, freezing, lyophilic drying, and control of thevaccine. The strain Trichophyton equinum No. 1-2251/70 (USSR) can beused as the immunogenic culture of Trichophyton equinum. This strain isdeposited at the All-Union Institute of Experimental Veterinary ofMoscow, 109472, USSR and is available on request. The strain has thefollowing cultural, morphological, and biological properties:

Cultural properties: A stab culture grown on wort-agar (pH 6.2-6.8) at atemperature of 26°-28° C. gives rapidly developing colonies (sometimeswith only slightly noticeable pleats). The diameter of 20-day-old colonyis 3-4 times greater than with the field strain.

A suspension culture grown on wort-agar for 5-7 days, forms a snow-whitefilm on the entire surface of the agar. The film is the mass of micro-and macroconidia.

Morphological properties: Ample micro- and macroconidia develop onwort-agar. Macroconidia are rudimentary, consisting mainly of 2-3segments; chlamydospores are only incidental, and are practicallyabsent. Owing to the presence of the mass of micro- and macroconidia,the washings of the culture with physiological saline solution ordistilled water is a thick milky suspension.

Antigenic properties: The titre of the sera of immune animals (rabbit,horses) is 1:640-1:1280 (agglutination test).

Virulent properties: When infected, the laboratory animals (guinea pigs,rabbits) and young horses, developed small foci (3-5 cm in diameter) onthe 7-15th day. The clinic is non-manifest. In 10-20 days, the animalsrecover without medicamentous treatment. The injection of live antigento healthy horses does not produce clinical signs of trichophytosis.

Immunogenic properties: Immunization of young horses produces activeimmunity in them that can last for at least five years. Administrationof live antigens to them produces proliferation of immunologicallycompetent cells. The character of the reaction is first plasmoblasticand then plasmocytary. The nutrient medium for growing the fungus iswort-agar, or any other nutrient medium that ensures easy growth of thefungal mass without loss of the immunogenic activity.

The inoculum is prepared by suspending lyophilically dried culture ofthe fungus in saline solution. The resulting material is used toinoculate matrices containing nutrient medium. The cultivation continuesfor 15 to 25 days at a temperature of 26° to 28° C.

The fungal mass is removed from the surface of the nutrient medium andtransferred into a homogenizer. Sterile water is then added and thesuspension is homogenized. The suspension is filled into vials, frozen,and dried lyophilically. It is recommended that the concentration of theobtained suspension, before lyophilic drying, should be adjusted to 200to 400 million microconidia in 1 ml of a drying medium. It is alsorecommended that a solution consisting of 35 to 45 percent by weight ofsaccharose and 5 to 7 percent by weight of gelatin in distilled watershould be used as the drying medium. Any other medium that would ensuresurvival of the required number of viable cells after lyophilic dryingcan be used as the drying medium. The finished vaccine is tested for theabsence of microflora, for the concentration of live fungus cells,harmlessness, and immunogenic potency.

For a better understanding of the invention, the following examples ofits practical embodiment are given by way of illustration.

EXAMPLE 1

Testing and Using the Vaccine.

The vaccine was tested on small laboratory animals and horses.

The experiments, carried out on 1-4 month old animals, showed thatactive immunity is formed in them in 14-30 days.

The duration and active immunity were studied on 79 foals which wereinfected with virulent strains of Trichophyton 1, 2, 3, 4, 8, 12 monthsand 1, 3, 4, and 5 years after vaccination. Controls were healthy,non-immunized animals selected on the analog basis.

The vaccinated animals resisted the infection while typicaltrichophytosis foci developed in all non-vaccinated animals.

When kept together with horses affected by trichophytosis, thevaccinated animals remained unaffected for five years. The vaccinatedhorses manifested marked stability to various epizootic strains of thecausative agents of trichophytosis in the horse. Commission tests of theinventive vaccine were carried out on 32 animals 6-9 months old. Theanimals were vaccinated and the controls were kept in the same stableswithout treatment. One month following the vaccination, the experimentalfoals were tested for stability against trichophytosis infection.Trichophyton equinum and Trichophyton mentagrophytes fungi were rubbedinto scarified skin of the animals.

The test showed that 95 percent of the vaccinated animals manifested amarked resistance of the experimental infection with virulent culturesof the dermatophytes. The clear clinical picture of trichophytosis andappearance of the secondary foci of infection were observed with 100percent of nonvaccinated animals (controls).

The next stage of the test was trial of the prophylactic efficacy of thevaccine. All newborn foals were vaccinated at a stud wheretrichophytosis had nested for years. During five years of observation,trichophytosis developed in only 3 out of 280 vaccinated foals. The studbecame completely safe with respect to trichophytosis.

Similar measures were taken at one of the central hippodromes, where allthe animals, and also all newly accepted horses were vaccinated. Thetotal number of vaccinated horses was over three thousand animals ofvarious breeds. Observation of the vaccinated animals showed that therewas not a single case of trichophytosis among them.

The vaccine was then tried at studs, hippodromes, and sports schools, atwhich trichophytosis was reported. The total number of vaccinated horseswas 20,000. This prevented new outbreaks of trichophytosis, and removedlimitations from sales and sporting contests. The expenditures formedicamentous treatment of animals with trichophytosis and disinfectionmeasures were thus abolished.

EXAMPLE 2

Preparing 25,000 doses of the vaccine.

The nutrient medium for growing the culture of the fungus Trichophytonequinum for the manufacture of vaccine is wort-agar. Malt-free,sterilized beer must is diluted with pure tap water to a carbohydratecontent of 7 percent (after Balling) and a pH of 6.8 to 7.0. Now 2.5-3percent by weight of agar-agar is added, the mixture is heated todissolve the agar, and filled into 1.3-1.5 liter matrices, 300 ml of themedium in each matrix. The medium is sterilized for 40 minutes under0.5-0.7 atmosphere pressure. The pH of the sterilized medium is usually6.4-6.8.

The seeding material is prepared as follows: The contents of an ampoule(3-5 ml) containing dried suspension of the strain Trichophyton equinumare resuspended in 50 ml of sterile saline solution. The thus-obtainedsuspension is kept at room temperature for an hour, then taken into aMohr pipette (or through a siphon), and used to inoculate the wort-agarmedium in the matrices. The quantity of the inoculum is 5-7 ml of thefungus suspension per matrix. The growth of the culture can be observedvisually as early as on the 3-5th day. The cultivation is continued for15-25 days. To prepare 25,000 doses of the vaccine, 50-100 matrices withgrown culture of the immunogenic strain are required. All operations arecarried out in a sterile box. The matrices with the culture are openedover the flame of a spirit burner. The biomass is taken from the surfaceof the nutrient medium and transferred into sterile Petri dishes. Thebiomass is then homogenized mechanically with distilled water (300-400ml of distilled water for the biomass taken from 5-10 matrices). Thesterile homogenizer is thereafter closed. To prevent contamination, theentire process of collecting the biomass and its transfer into thehomogenizer should be carried out with a burning spirit burner. Thebiomass is homogenized for 15-20 minutes until a suspension of separatedcells is obtained.

The homogenized fungal biomass is then transferred into flasks andsaccharose-gelatin solution is added to attain the concentration ofmicro- and macroconidia of 200-400 million in 1 ml.

This saccharose-gelatin solution is used as a protective medium fordrying. The solution is prepared by adding 4-8 percent by weight ofgelatin into hot distilled water, and then (after dissolution), 10-30percent by weight of saccharose (calculations with reference to thewater taken) is added. The pH of the solution is adjusted to 7.0-7.4.The solution is then sterilized with steam for three days by 30 minutesessions, or under pressure of 0.5-0.7 atmosphere for 40 minutes. Aftersterilization, the pH of the solution is within 6.2-6.8.

The prepared suspension of cells is filled into sterile vials, 2 ml intoeach vial of 15-20 ml capacity. The vials prepared for drying are placedinto refrigerating chambers at temperatures from -50° to -60° C. for10-20 hours. The vials are then placed into sublimation dryingapparatus. The drying is continued for 40-60 hours. In 1-3 hours, assoon as the vacuum of 9-10 mm Hg is attained, the vials are heated to25°-40° C., and in 10-15 hours the temperature on the shelves of thesublimation apparatus is adjusted to 20°-25° C. The residual moisturecontent in the apparatus atmosphere should be not below 1 and not above3 percent. The vaccine is then checked for solubility, residual moisturecontent, purity with respect to bacteria and moulds, concentration ofmicroconidia, the quantity of viable cells, harmlessness, andimmunogenic potency.

To determine the solubility of the vaccine, 10 ml of physiologicalsaline solution are added into a vial containing dry vaccine. The vialcontents are shaken: the vaccine should dissolve completely in 2-3minutes.

The vaccine is tested for the absence of extraneous flora by seeding itat random onto nutrient media. Meat-peptone agar, meat-peptone broth, orthe Kitt-Tarozzi medium are used for detection of extraneous bacterialflora, and Czapek medium and wort-agar are used to detect extraneousfungi. The test tubes are kept for ten days in thermostats at atemperature of +37° C. and 26° C. All tests must show the absence of anyextraneous microflora. Residual moisture content is determined by dryingthe vaccine under atmospheric pressure at a temperature of 100°-105° C.for an hour by accepted methods.

The concentration of micro- and macroconidia is determined by countingthem in a special chamber for calculation of formed blood elements(Goriaev's chamber, for example).

The viability of the fungus cells is determined by suspending thevaccine in a sterile saline solution at a pH of 6.2-7.0 to restore theoriginal volume. (If the vaccine was filled into vials in 2 ml portions,2 ml of the saline solution should be added, if 4 ml--4 ml sould beadded, etc.). A series of solutions having different concentrations from10⁻¹ to 10⁻⁸ is then prepared. The material from the 6th, 7th and the8th test tubes is seeded onto wort-agar. To determine the viability ofcells, the number of survivals in 1 ml of the vaccine in the last twosolutions in the series in which the fungus cells grow is counted.

The vaccine is tested for harmlessness and immunogenic potency onrabbits, guinea pigs, or on foals. The rabbits are given 1 ml and guineapigs 0.3-0.5 ml of the vaccine intramuscularly. The vaccine should be inthe suspension form having the proper operative concentration.

The animals are observed for ten days. All rabbits or guinea pigs mustsurvive. The batches of vaccine that kill a single animal or producecomplications on the injection site (abscesses, necrosis of the tissue),should be tested again on a doubled number of animals and checked alsofor the presence of extraneous flora.

If a single animal perishes or extraneous microflora is discovered inthe batch, it should be rejected and acknowledged as unfit forvaccination.

The immunogenic activity of the vaccine is tested on 7-10 month oldfoals. The criteria of activity are (a) the number of viable cells in 1ml of the vaccine being not less than 200-300 million microconidia, and(b) appearance of local reaction to vaccination on foals that ismanifested by painful edema at the injection site. The edema shouldappear on the 1-2nd day following the vaccination and should resolve in3-5 days. The vaccine intended for use should be kept in a refrigeratorat a temperature from 4° to 12° C.

The working solution should be prepared by dissolving the dry vaccine insterile saline solution having a pH 6.2-7.0 (1 ml of the dry suspensiondissolved in 5 ml of saline solution). The concentration of themicroconidia in the thus-prepared vaccine is 30-45 million microconidiain 1 ml of sterile physiological saline solution.

What is claimed is:
 1. A vaccine for prophylaxis of trichophytosis in the horse, comprising a suspension of microconidia of the immunogenic strain No. L-2251/70 (USSR) of Trichophyton equinum in sterile physiological salt solution at a pH of 6.2 to 7.0, the concentration of the microconidia ranging from 30 to 45 million per 1 milliliter of the sterile physiological salt solution.
 2. A method for preparing the vaccine for prophylaxis of trichophytosis in the horse defined by claim 1, comprising growing the immunogenic culture of Trichophyton equinum on a solid nutrient medium at a temperature of 26° to 28° C. for 15 to 25 days until the optimum amount of microconidia of said culture has accumulated, separating the resulting propagated biomass, homogenizing the biomass to prepare a suspension of separate cells of the microorganisms, and drying the suspension thus obtained to isolate the end product.
 3. The method according to claim 2, in which, prior to the drying, the concentration of the suspension is adjusted to 200 to 400 million microconidia per 1 milliliter of a drying medium.
 4. The method according to claim 3, in which the drying medium is a solution consisting of 35 to 45 percent by weight of saccharose and 5 to 7 percent by weight of gelatin in distilled water.
 5. The method of claim 2 in which the nutrient medium is wort-agar.
 6. A method of vaccinating a trichophytosis-free horse to immunize it against trichophytosis, comprising injecting a first dose of 1 to 2 milliliters of the vaccine defined by claim 1 into an injection site in the third middle part of the neck of the horse, allowing an interval of 10 to 14 days to elapse, and injecting a second dose of 1 to 2 milliliters of said vaccine into an injection site in said third middle part of the neck of the horse.
 7. A method of treating a trichophytosis-infected horse and thereafter conferring immunity against said disease in said horse, comprising injecting a first dose of 2 to 4 milliliters of the vaccine defined by claim 1 into an injection site in the third middle part of the neck of the infected horse, allowing an interval of 10 to 14 days to elapse, and injecting a second dose of 2 to 4 milliliters of said vaccine into an injection site in said third middle part of the neck of the infected horse.
 8. The method according to claim 2 wherein the suspension of separate cells of the micro-organisms is lyophilically dried.
 9. The method according to claim 2 wherein the nutrient medium is wort-agar, and the suspension of separate cells of the micro-organisms is lyophilically dried. 